The present invention is concerned with the stereoisomeric forms of itraconazole and saperconazole, processes for preparing said stereoisomeric forms, the complexes thereof with cyclodextrin derivatives, pharmaceutical compositions comprising said complexes and methods of preparing said complexes and pharmaceutical compositions.
Itraconazole or (xc2x1)-cis-4-[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinylphenyl]-2,4-dihydro-2-(1-methyl-propyl)-3H-1,2,4-triazol-3-one, is a broadspectrum antifungal compound developed for oral, parenteral and topical use and is disclosed in U.S. Pat. No. 4,267,179. Its difluoro analog, saperconazole or (xc2x1)-cis-4-[4-[4-[4-[[2-(2,4-difluorophenyl)-2-(1H-2,4-triazol-1-yl-methyl)-1,3-dioxolan-4-yl]methoxylphenyl]-1-piperazinyl]-phenyl]-2,4-dihydro-2-(1-methylpropyl)-3H-1,2,4-triazol-3-one, has improved activity against Aspergillus spp. and is disclosed in U.S. Pat. No. 4,916,134. Both compounds exist as a mixture of four stereoisomers.
The development of efficaceous pharmaceutical compositions of itraconazole and saperconazole is hampered considerably by the fact that said compounds are only very sparingly soluble in water. The solubility of both compounds can be increased by complexation with cyclodextrins or derivatives thereof as described in WO 85/02767 and U.S. Pat. No. 4,764,604.
Unexpectedly, it has now been found that each of the individual stereoisomers of itraconazole and saperconazole have greater water solubility than the diastereomeric mixtures of said compounds, in particular when complexed with cyclodextrin or its derivatives. As a result, pharmaceutical compositions having good bioavailability, yet comprising less cyclodextrin as a complexing agent, can be prepared.
The present invention is concerned with the stereoisomeric forms of itraconazole (X=Cl) and saperconazole (X=F), which may be represented by the formula 
and the pharmaceutically acceptable acid addition salt forms thereof. The three asterisks indicate the three chiral centers, and xe2x80x98cisxe2x80x99 means that the (1H-1,2,4triazol-1-ylmethyl) moiety and the substituted phenoxy moiety are located at the same side of the plane defined by the 1,3Aioxolane ring.
The four possible stereoisomeric cis forms can be described using various rules of nomenclature. The following tables show the correlation among the C.A. stereochemical descriptor, the absolute configuration at each of the chiral centers and the specific optical rotation [xcex1]D20 in 1% methanol (itraconazole; table I) (saperconazole; table II).
The term xe2x80x98stereoisomeric formxe2x80x99 as used herein concerns compounds having a stereoisomeric purity of at least 96% up to a stereoisomeric purity of 100%, in particular compounds having a stereoisomeric purity of 98% up to 100%. In particular, said stereoisomeric forms define compounds having an enantiomeric excess and a diastereomeric excess of at least 96% up to 100%, more particularly, having an enantiomeric excess and a diastereomeric excess of 98% up to 100%.
With regard to the intermediates described hereinafer, the term xe2x80x9cenantiomerically purexe2x80x9d defines intermediates having an enantiomeric excess of at least 96% up to 100%, more particularly, having an enantiomeric excess of 98% up to 100%.
The stereoisomeric forms of the compounds of formula (I) have basic properties. The pharmaceutically acceptable acid addition salts as mentioned hereinabove comprise the therapeutically active non-toxic acid addition salt forms which the compounds of formula (I) are able to form. Said salt forms can conveniently be obtained by treating the base form of the compounds of formula (I) with appropriate acids such as inorganic acids, for example, hydrohalic acid, e.g. hydrochloric, hydrobromic and the like acids, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids, such as, for example, acetic, propanoic, hydroxyacetic, 2-hydroxypropanoic, 2-oxopropanoic, ethanedioic, propanedioic, butanedioic, (Z)-2-butenedioic, (E)-2-butenedioic, 2-hydroxybutanedioic, 2,3-dihydroxybutanedioic, 2-hydroxy-1,2,3-propanetricarboxylic, methanesulfonic, ethanesulfonic, benzenesulfonic, 4-methylbenzenesulfonic, cyclohexanesulfamic, 2-hydroxybenzoic, 4-amino-2-hydroxybenzoic and the like acids. Conversely the salt form can be converted by treatment with alkali into the free base form.
The term acid addition salt also comprises the hydrates and solvent addition forms which the compounds of formula (I) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.
The four individual stereoisomeric forms of the compounds of formula (I) can be prepared by O-alkylating an enantiomerically pure phenol of formula (xe2x88x92)-(R)-(II) or (+)-(S)-(II) with an enantiomerically pure 1,3-dioxolane derivative of formula (xe2x88x92)-(2S,cis)-(III) or (+)-(2R,cis)-(III) wherein xe2x80x94OR represents a sulfonyloxy leaving group such as 4-methylbenzenesulfonyloxy (tosylate) or methanesulfonyloxy (mesylate). 
Said O-alkylation reaction can conveniently be conducted following art-known procedures, e.g. by stirring and heating the reactants in an appropriate solvent such as a dipolar aprotic solvent, e.g. N,N-dimethylformamide, N,N-dimethylacetamide and the like, in the presence of a base such as, an alkali metal hydroxide, e.g. sodium or potassium hydroxide. The thus obtained stereoisomeric forms of the compound of formula cis-(I) may be purified further following art-known methodologies such as liquid chromatography and crystallization.
The relation between the stereochemistry of the stereoisomeric forms of the compound of formula (I) obtained in said O-alkylation reaction and the stereochemistry of the starting materials (II) and (III) is shown in the table herebelow.
The enantiomerically pure phenol of formula (xe2x88x92)-(R)-(II) can conveniently be prepared starting from (S)-2-butanol (IV). The enantiomeric (S)-butanol (IV) can be converted into a corresponding (S)-sulfonate (V) by reaction with 4-methylbenznesulfonyl chloride (R=Me) or 1-bromo-4-benzenesulfonyl chloride (R=Br) in pyridine. 
Enantioselective coupling of the sulfonate (S)-(V) with the triazolone (VI) (prepared as described in Example XVII of U.S. Pat. No. 4,267,179) proceeds with inversion of configuration at the chiral center and yields (xe2x88x92)-(R)-(VII). 
Said coupling reaction can be conducted in a reaction inert solvent such as a dipolar aprotic solvent, preferably N,N-dimethylformamide, in the presence of a base such as sodium hydride. The enantiomeric excess of the thus obtained product (xe2x88x92)-(R)-(VII) ranges from about 65% to about 75% and can be increased up to an enantiomeric excess e.e. greater than 98% by converting (xe2x88x92)-(R)-(VII) into the (R)-camphor sulfonate salt in acetone, followed by repeated recrystallization of the salt from ethanol/acetone mixtures (2:7; v/v).
Dealkylation of the thus purified anisole (xe2x88x92)-(R)-(VII) by heating to reflux in concentrated hydrobromic acid yields the enantiomerically pure phenol (xe2x88x92)-(R)-(II). In order to avoid bromination in the last mentioned step, it is advantageous to add sodium sulfite to the reaction mixture. 
The other enantiomeric form, (+)-(S)-(II), can be prepared in a similar manner starting from (R)-2-butanol. Obviously, the optical purification of the thus obtained intermediate product (+)-(S)-(VII) is conducted on the (S)-camphor sulfonate salt.
An alternative procedure for preparing (xe2x88x92)-(R)-(VII) and (+)-(S)-VII) comprises resolving the corresponding racemate (xc2x1)-(VII) by fractional crystallization with enantiomerically pure camphor sulfonic acid from a mixture of ethanol/acetone (1:4; v/v). Further optical purification to e.e.  greater than 98% can be achieved by repeatedly recrystallizing the camphor sulfonate salt as described hereinbefore. (xe2x88x92)-(R)-(VII) is obtained from recrystallizing the racemate (xc2x1)-(VII) with (R)-camphor sulfonic acid; (+)-(S)-(VII) can be obtained similarly with (S)-camphor sulfonic acid from racemic (xc2x1)-(VII), or preferably from the mother liquors of the previous resolution step with (R)-camphor sulfonic acid which are enriched in the (+)-(S)-(VII) enantiomer.
Preferably, however, the racemate (xc2x1)-(VII) can be resolved by liquid chromatography using a chiral stationary phase such as an amylose derivative, in particular amylose tris-(3,5-dimethylphenyl) carbamate coated on a macroporous xcex3-aminopropyl silica matrix (Chiralpak AD(trademark), Daicel), or a Pirkle type stationary phase. The separation is preferably conducted with an alcohol such as methanol or ethanol (optionally denatured with 1% methanol) as the mobile phase. In order to speed up the separation, the elution chromatography process may be conducted at a temperature higher than ambient (about 30xc2x0 C.). More time can yet be gained by using recycling peak shaving technology. For larger scale applications, the full continuous simulated moving bed adsorption technology can advantageously be used.
In an identical manner as described in the preceding paragraph, the racemate (xc2x1)-(II) can be separated into its two enatiomers on amylose tris-(3,5-dimethylphenyl) carbamate coated on a macroporous xcex3-aminopropyl silica matrix (Chiralpak AD(trademark), Daicel).
The enantiomerically pure intermediate (+)-(2R,cis) (III) wherein xe2x80x94OR is tosylate, can be prepared from the commercially available (S)-2,2dimethyl-1,3-dioxolane-4-yl methanol (S)-(VIII). Reaction of (S)-(VIII) with 4-methylbenzenesulfonyl choride in pyridine and hydrolysis of the thus obtained product in an aqueous acid solution, e.g. hydrochloric acid 6N, optionally in admixture with a solvent such as an alcohol or a ketone, e.g. acetone, yields the corresponding (R)-1,2-dihydroxypropyl tosylate (IX). 
Acetalation of (R)-(IX) with 1-(2,4dihalophenyl)-2-(1H-1,2,4-triazolyl)ethanone (X) under mild conditions yields a cis/trans mixture of dioxolanes from which the cis isomer (+)-(2R,cis)-(III) is obtained after chromatography. 
Said acetalation reaction can conveniently be conducted by stirring and heating the reagents in a reaction-inert solvent, such as a halogenated hydrocarbon, e.g. dichloromethane or trichloromethane, in the presence of an acid, preferably a sulfonic acid, such as methanesulfonic acid.
In a similar manner, starting from (R)-2,2-dimethyl-1,3-dioxolane-4-yl methanol (R)-(VIII) there can be prepared (xe2x88x92)-(2S,cis)-(III).
The racemic compounds of formula (xc2x1)-cis(III) 
wherein X represents chloro or fluoro and R represents 4-methylbenzenesulfonyl, methanesulfonyl, but also C1-5alkylcarbonyl can conveniently be separated into their individual enantiomers by liquid chromatography using a chiral stationary phase such as a cellulose or amylose derived stationary phase. A preferred example of such a phase is cellulose tris-(4-methylbenzoate) either in its pure polymeric form or coated on a macroporous xcex3-aminopropyl silica matrix (Chiralcel OD(trademark), Daicel). The mobile phase preferably is an alcohol such as methanol or ethanol (optionally denatured with 1% methanol). Methanol is preferred with the cellulose derivative in the pure polymeric form. In order to speed up the separation, the elution chromatography process may be conducted at a temperature higher than ambient (about 30xc2x0 C.). More time can yet be gained by applying recycling peak shaving technology. For larger scale operations, the elution chromatography process is preferably replaced by full continuous simulated moving bed adsorption technology.
In case R represents C1-5alkylcarbonyl, the separated enantiomers are converted into the mesylate or tosylate derivatives following art-known procedures such as saponification and subsequently sulfonylation.
The individual stereoisomeric forms of itraconazole and saperconazole have antifungal properties. Each of the four isomers contributes to the overall activity of the parent compound and none appears to be more active than said parent compound The (xe2x88x92)-[2S-[2xcex1,4xcex1,4(R)]] and (xe2x88x92)-[2S-[2xcex1,4xcex1,4(S)]] isomers however appear more potent than their respective enantiomorphs.
The individual stereoisomers of itraconazole and saperconazole can also be obtained by separating the diastereomeric mixture by liquid chromatography on a chiral stationary phase such as an amylose derivative, in particular amylose tris-(3,5-dimethylphenyl) carbamate coated on a macroporous aminopropyl silica matrix (Chiralpak AD(trademark), Daicel). Suitable mobile phases are alcohols in particular methanol and ethanol (optionally denatured with 1% methanol). In order to speed up the process and keep the product into solution, the separation is preferably performed at a temperature above ambient (up to 50xc2x0 C.). Limitations to the separation process are the fact that the compounds are poorly soluble and only small amounts can be processed at one time. Furthermore, the occurrence of 4 peaks precludes the use of recycling and peak-shaving technology. Consequently, separation of the intermediates for preparing stereochemically pure itraconazole and saperconazole is to be preferred over the separation of the end-products.
The individual stereoisomeric forms of itraconazole and saperconazole have greater water solubility than the diastereomeric mixtures of said compounds particularly when the individual stereoisomers are complexed with a cyclodextrin derivative.
Appropriate cyclodextrin derivatives are xcex1-,xcex2-, xcex3-cyclodextrins or ethers and mixed ethers thereof wherein one or more of the hydroxy groups of the anhydroglucose units of the cyclodextrin are substituted with C1-6alkyl, particularly methyl, ethyl or isopropyl; hydroxyC1-6alkyl, particularly hydroxyethyl, hydroxypropyl or hydroxybutyl; carboxyC1-6alkyl, particularly carboxymethyl or carboxyethyl; C1-6alkyl-carbonyl, particularly acetyl; C1-6alkyloxycarbonylC1-6aLkyl or carboxyC1-6alkyloxyC1-6alkyl, particularly carboxymethoxypropyl or carboxyethoxypropyl; C1-6alkylcarbonyloxyC1-6alkyl, particularly 2-acetyloxypropyl. Especially noteworthy as complexants and/or solubilizers are xcex2-CD, 2,6-dimethyl-xcex2-CD, 2-hydroxyethyl-xcex2-CD, 2-hydroxyethyl-xcex3-CD, 2-hydroxypropyl-xcex3-CD and (2-carboxymethoxy)propyl-xcex3-CD, and in particular 2-hydroxypropyl-xcex2-CD.
The term mixed ether denotes cyclodextfin derivatives wherein at least two cyclodextrin hydroxy groups are etherified with different groups such as, for example, hydroxypropyl and hydroxyethyl.
The average molar substitution (M.S.) is used as a measure of the average number of moles of alkoxy units per mole of anhydroulucose. In the cyclodextrin derivatives for use in the compositions according to the present invention the M.S. is in the range of 0.125 to 10, in particular of 0.3 to 3, or from 0.3 to 1.5. Preferably the M.S. ranges from about 0.3 to about 0.8, in particular from about 0.35 to about 0.5 and most particularly is about 0.4.
The average substitution degree (D.S.) refers to the average number of substituted hydroxyls per anhydroglucose unit. In the cyclodextrin derivatives for use in the compositions according to the present invention the D.S. is in the range of 0.125 to 3, in particular of 0.2 to 2 or from 0.2 to 1.5. Preferably the D.S. ranges from about 0.2 to about 0.7, in particular from about 0.35 to about 0.5 and most particularly is about 0.4.
More particular xcex2- and xcex3-cyclodextrin hydroxyalkyl derivatives for use in the compositions according to the present invention are partially substituted cyclodextrin derivatives wherein the average degree of alkylation at hydroxyl groups of different positions of the anhydroglucose units is about 0% to 20% for the 3 position, 2% to 70% for the 2 position and about 5% to 90% for the 6 position. Preferably the amount of unsubstituted, xcex2- or xcex3-cyclodextrin is less than 5% of the total cyclodextrin content and in particular is less than 1.5%. Another particularly interesting cyclodextrin derivative is randomly methylated xcex2-cyclodextrin.
Most preferred cyclodextrin derivatives for use in the present invention are those partially substituted xcex2-clodextrin ethers or mixed ethers having hydroxypropyl, hydroxyethyl and in particular 2-hydroxypropyl and/or 2-(1-hydroxypropyl) substituents.
The most preferred cyclodextrin derivative for use in the compositions of the present invention is hydroxypropyl-xcex2-cyclodextrin having a M.S. in the range of from 0.35 to 0.50 and containing less than 1.5% unsubstituted xcex2-cyclodextrin.
Substituted cyclodextrins can be prepared according to procedures described in U.S. Pat. No. 3,459,731, EP-A-0,149,197, EP-A-0,197,571, U.S. Pat. No. 4,535,152, WO-90/12035 and GB-2,189,245.
Other references describing cyclodextrins for use in the compositions according to the present invention, and which provide a guide for the preparation, purification and analysis of cyclodextrins include the following: xe2x80x9cCyclodextrin Technologyxe2x80x9d by Jxc3x3zsef Szejtli, Kluwer Academic Publishers (1988) in the chapter Cyclodextrins in Pharmaceuticals; xe2x80x9cCyclodextrin Chemistryxe2x80x9d by M. L. Bender et al., Springer-Verlag, Berlin (1978); xe2x80x9cAdvances in Carbohydrate Chemistryxe2x80x9d, Vol. 12 Ed. by M. L. Wolfrom, Academic Press, New York (157) in the chapter The Schardinger Dextrins by Dexter French at p. 189-260; xe2x80x9cCyclodextrins and their Inclusions Complexesxe2x80x9d by J. Szejtli, Akademiai Kiado, Budapest, Hungary (1982); I. Tabushi in Acc. Chem. Research, 1982, 15, p. 66-72; W. Sanger, Angewandte Chemie, 92, p. 343-361 (1981); A. P. Croft and R. A. Bartsch in Tetrahedron, 39, p. 1417-1474 (1983); Irie et al. Pharmaceutical Research, 5 p. 713-716, (1988); Pitha et al. Int. J. Pharm. 29, 73, (1986); DE 3118218; DE 3,317,064; EP-A-94,157; U.S. Pat. Nos. 4,659,696; and U.S. Pat. No. 4,383,992. Particular attention should be paid to those references which describe the preparation and purification methods which provide cyclodextrin mixtures wherein the amount of unreacted cyclodextrin is less than 5% of the total cyclodextrin content.
The complexes of the individual stereoisomeric compounds of formula (I) with cyclodextrin derivatives as mentioned hereinabove, can conveniently be prepared by dissolving the cyclodextrin or ether derivative thereof in water and adding thereto the individual stereoisomeric compound of formula (I) while stirring or shaking the resulting mixture until complete dissolution is obtained. For storage purposes it may be advantageous to dehydrate the thus obtained solutions, e.g. by freeze-drying or spray-drying. The dehydrated complexes can easily be reconstituted by the addition of water or an aqueous cyclodextrin solution.
The complexes of the individual stereoisomeric compounds of formula (I) with cyclodextrin derivatives are particularly useful for the preparation of pharmaceutical compositions for oral, parenteral or topical administration. Said pharmaceutical compositions comprise as active ingredient a complex as defined hereinabove and a pharmaceutically acceptable carrier. Said carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for administration orally, rectally, percutaneously, or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause a significant deleterious effect to the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermnal patch, as a spot-on, as an ointment. Acid addition salts of (I) due to their increased water solubility over the corresponding base form, are obviously more suitable in the preparation of aqueous compositions. It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
In the final compositions, the cyclodextrin will comprise about 2.5 to 20% by weight, in particular about 5 to 20%, more in particular 5 to 15%, for example about 10%, with the remainder being water, the active ingredient and any excipients.
In particular, stable pharmaceutical compositions may consist of water, cyclodextrin and the active ingredient alone without the need of additional stabilizers such as, human serum albumin, bovine serum albumin, lecithin, methyl cellulose, polyethylene glycol, sulfur containing reducing agents, urea, amino acids and surfactants. There may be added a pH-adjusting agent e.g. hydrochloric acid, acetic acid, citric acid, sodium hydroxide, potassium hydroxide or a salt of any of these, in particular sodium citrate. The appropriate pH for formulating the itraconazole or saperconazole stereoisomers ranges from 6.5 to 7.4, in particular from 6.8 to 7.0. Appropriate preservatives for the above pharmaceutical preparations are: alcohols, for example, ethanol, 1,3-propanediol, benzylalcohol or derivatives thereof, phenyl ethyl alcohol, phenol or phenol derivatives such as butylparaben, methylparaben, m-cresol or chlorocresol; acids, for example, benzoic acid, sorbic acid, citric acid, sodium propionate, EDTA disodium; chlorhexidine; hexamidine diisetionate; hexetidine; optionally in combination with sodium bisulfite, or with propyleneglycol, or less preferably quaternary ammonium salts, metallic compounds such as zinc oxide, thiomersal and phenyl mercury salts, e.g. phenylmercuric acetate
For the preparation of an injectable it is appropriate to add an isotonizing agent, e.g. sodium chloride, potassium chloride, sorbitol.
It may further be appropriate to add a suitable complexing agent such as calcium chloride, citrate, EDTA and the like pharmaceutically acceptable metal ion complexing agents. For example, there may be added calcium chloride at a concentration of about 0.02-2 g/l.
Said compositions can conveniently be prepared by dissolving the cyclodextrin or ether derivative thereof in water and adding thereto the compound of formula (I) as well as other adjuvants and components such as, for example, sodium chloride, potassium nitrate, glucose, mannitol, sorbitol, xylitol and buffers such as, for example, phosphate, acetate or citrate buffer, and optionally concentrating or drying the solution by evaporation under reduced pressure or by lyophilization; and further optionally reconstituting the lyophilized residue with water.
Preferred preparations according to the present invention have low toxicity, and are not irritating, thus permitting the manufacture of an injectable medicament which may safely be used in repeated dose regimes without the risk of immunogenic reactions.
The aqueous preparations according to invention, and an excipient if required, may also be freeze-dried or spray-dried following art-known procedures to give a dehydrated composition which may be stored for a long period of time and dissolved before administration. In said freeze-dried or spray-dried formulations the molar ratio and the weight-to-weight ratio of cyclodextrin to active ingredient may be the same as in the above-mentioned aqueous solutions. As it is convenient in a number of instances to reconstitute said freeze-dried or spray-dried formulation in an aqueous cyclodextrin solution, the molar ratio and the weight to weight ratio of cyclodextrin to active ingredient may also be lower than in the above mentioned aqueous solutions.